Biochimica et Biophysica Acta 1791 (2009) 719–729

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Biochimica et Biophysica Acta

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First evidence for the salt-dependent folding and activity of an esterase from thehalophilic archaea Haloarcula marismortui

Marcelo Müller-Santos a, Emanuel M. de Souza a, Fabio de O. Pedrosa a, David Alexander Mitchell a,Sonia Longhi b, Frédéric Carrière c,⁎, Stéphane Canaan c,⁎, Nadia Krieger d,⁎a Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná,Cx.P. 19046 Centro Politécnico, Curitiba 81531-980, Paraná, Brazilb Laboratoire d'Architecture et Fonction des Macromolecules Biologiques, UMR6098, CNRS, Université de Provence Université de la Méditerrané,, 163 Avenue de Luminy,13288 Marseille cedex 9, Francec CNRS Laboratoire d'Enzymologie Interfaciale et de Physiologie de la Lipolyse, UPR9025, 31 Chemin Joseph-Aiguier, F-13402 Marseille cedex 20, Franced Laboratório de Tecnologia Enzimática e Biocatálise, Departamento de Química, Universidade Federal do Paraná, Cx.P. 19081 Centro Politécnico, Curitiba 81531-980, Paraná, Brazil

Abbreviations: Af EST, Archeoglobus fulgidus esterase;dimethyl sulfoxide; E600, diethyl-p-nitrophenyl phoacidocaldarius esterase; HCA, hydrophobic cluster anaHaloarcula marismortui; HMM, hidden Markov model;HSL, hormone sensitive lipase; IMAC, Immobilized Mgraphy; Lipase inhibitor 7600, 5-methoxy-3-(4-phenox2-one; MALDI-TOF, Matrix-assisted laser desorption/iophenylmethylsulphonyl fluoride, SDS-PAGE sodium dgel electrophoresis; Sso EST, Sulfolobus solfataricuchromatography⁎ Corresponding authors. S. Canaan is to be contac

France. Tel.: +33 4 91 16 44 93; fax: +33 4 91 71Department, Federal University of Parana, Centro PolitecCuritiba, PR, Brazil. Tel.: +55 41 33613470; fax: +55 41

E-mail addresses: carriere@ibsm.cnrs-mrs.fr (F. Carristephane.canaan@ibsm.cnrs-mrs.fr (S. Canaan), nkriege

1388-1981/$ – see front matter © 2009 Elsevier B.V. Adoi:10.1016/j.bbalip.2009.03.006

a b s t r a c t

a r t i c l e i n f o

Article history:Received 27 November 2008Received in revised form 3 March 2009Accepted 5 March 2009Available online 17 March 2009

Keywords:EnzymeEsteraseHaloarchaeaHaloadaptationHormone-sensitive lipaseProtein folding

A gene encoding an esterase from Haloarcula marismortui, a halophilic archaea from the Dead Sea, wascloned, expressed in Escherichia coli, and the recombinant protein (Hm EST) was biochemically characterized.The enzymatic activity of Hm EST was shown to exhibit salt dependence through salt-dependent folding. HmEST exhibits a preference for short chain fatty acids and monoesters. It is inhibited by phenylmethylsulfonylfluoride, diethyl-p-nitrophenyl phosphate, and 5-methoxy-3-(4-phenoxyphenyl)-3H-[1,3,4]oxadiazol-2-one,confirming the conclusion from sequence alignments that Hm EST is a serine carboxylesterase belonging tothe hormone-sensitive lipase family. The activity of Hm EST is optimum in the presence of 3 M KCl and noactivity was detected in the absence of salts. Far–UV circular dichroism showed that Hm EST is totallyunfolded in salt-free medium and secondary structure appears in the presence of 0.25–0.5 M KCl. After saltdepletion, the protein was able to recover 60% of its initial activity when 2 M KCl was added. A 3D model ofHm EST was built and its surface properties were analyzed, pointing to an enrichment in acidic residuesparalleled by a depletion in basic residues. This peculiar charge repartition at the protein surface supports abetter stability of the protein in a high salt environment.

© 2009 Elsevier B.V. All rights reserved.

1. Introduction

Esterases and lipases are important tools in biotechnology, wherethey find applications in the resolution of racemates and the synthesisof esters [1–3]. In these applications they may be subjected toaggressive conditions such as relatively high temperatures or thepresence of organic solvents. Various authors have suggested that

CD, circular dichroism; DMSO,sphate; EST2, Alicyclobacilluslysis; Hm EST, esterase fromHPL, human pancreatic lipase;etal Ion Affinity Chromato-yphenyl)-3H-[1,3,4]oxadiazol-nization–time of flight; PMSFodecyl sulfate polyacrylamides esterase; TLC, thin-layer

ted at CNRS-EIPL, Marseilles,58 57. N. Krieger, Chemistrynico, PO Box 19081, 81531-98033613186.ère),r@ufpr.br (N. Krieger).

ll rights reserved.

esterases and lipases from extremophiles may be active and stableunder such aggressive reaction conditions [4–7]. This has led naturallyto the search for these enzymes in Archaea, many of which inhabitextreme environments. In fact, several esterases and lipases havealready been characterized from thermophilic archaea and, asexpected, these enzymes have shown relatively high thermostabilities[8–12]. On the other hand, relatively little attention has been given toesterases and lipases from halophilic archaea.

Halophilic archaea live in very high salt concentrations [13]. Inorder to maintain an osmotic balance they accumulate high saltconcentrations within the cytoplasm [14,15]. As a result, not only theirextracellular enzymes, but also their intracellular enzymes, areadapted to operating in high salt concentrations [14–16]. Thisadaptation might confer stability in the low water environments inbiocatalytic reactions carried out in organic solvents.

Although previous work has focused on finding lipase activity inhalophilic archaea, there have not been attempts specifically aimed atfinding esterases. Lipolytic activity has been detected for isolates ofHalobacterium sp. on agar plates with Tween 20–80 in the presenceof calcium [17] and against olive oil with a crude preparation obtainedfrom a submerged culture of Natronococcus sp. [18]. However, theenzymes responsible for these activities have not yet been isolated

720 M. Müller-Santos et al. / Biochimica et Biophysica Acta 1791 (2009) 719–729

and characterized. This scarcity in biochemical data is to a large extentdue to difficulties in producing a sufficient amount of the enzymeduring cultivation of the archaea.

In the current work we circumvented these difficulties by usingheterologous expression, a strategy that has successfully been used forthe production of other enzymes from halophilic archaea [19]. The lipCgene identified from Haloarcula marismortui was cloned and over-expressed in Escherichia coli, and the recombinant protein (Hm EST)was purified and biochemically characterized. We particularlyinvestigated the effects of salts on the folding and activity of HmEST, seeking to obtain a better understanding of the structure–function relationships of halophilic esterases.

2. Materials and methods

2.1. Materials

Vinyl series, monocaprylin, dicaprylin, monocaprin, dicaprin,monolaurin, triacetin, tributyrin, tricaprylin, tricaprin and trioleinwere purchased from Sigma-Aldrich-Fluka (St. Louis, MO, USA).Monobutyrinwas from Acros (Geel, Belgium). Dibutyrinwas preparedat the laboratory by enzymatic hydrolysis of tributyrin with humanpancreatic lipase (HPL) and purification by preparative TLC. Pfx DNApolymerase and E. coli strain DH10B were purchased from Invitrogen(San Diego, CA, USA). E. coli strain BL21(DE3)pLysS and the plasmidpET14b were purchased from Novagen (San Diego, CA, USA). T4 ligaseand the restriction enzymes were purchased from Promega (Madison,WI, USA). The HiTrap Chelating Column (5 mL) and Octyl SepharoseCL-4B matrix were purchased from Amersham Biosciences (Uppsala,Sweden). The protein molecular mass markers were from Fermentas(St. Rémy Lès Chevreuse, France).

2.2. Strains and plasmids

The H. marismortui strain was a kind gift from Professor FranciscoRodríguez-Valera (Universidad Miguel Hernández, Alicante, Spain).The expression plasmid pET14b-lipC was a construction obtained byinsertion of the lipC gene from H. marismortui into pET14b. For theenzyme expression, E. coli BL21(DE3)pLysS was used.

2.3. Bioinformatic analysis

The bioinformatic analysis of the lipC gene was done using thefollowing tools. Multiple sequence alignment was performed usingthe ClustalW algorithm [20]. The amino acid sequences werecompared with the non-redundant sequence databases deposited atthe NCBI (National Center for Biotechnology Information, USA) usingthe BLAST algorithm [21]. The prediction of transmembrane domainswas performed using the Dense Alignment Surface (DAS) method[22]. The prediction of the signal peptide sequence was performedusing the SignalP 3.0 application [23]. Primary structure analysis wasperformed using the ExPASy Proteomics tools. The ProtParam tool wasused to calculate the theoretical parameters of the protein [24].

2.4. Structural modeling

Modeling was carried out using the SAM-T06 server that usesiterative hidden Markov model-based methods (HMMs) for con-structing protein family profiles, using only sequence information[25–29]. It is a fully automated method that generates a three-dimensional (3D) structural model by making use of a set ofhomologous structures. The SAM-T06 server uses a multi-stepprocedure consisting of 1) iterative search within the PDB data base[30] for sequences homologous to the query, using the SAM-T2K andSAM-T04 algorithms, 2) prediction of local structure properties usingneural networks, 3) identification of possible fold recognition

templates using two and three-track HMMs, 4) generation ofalignments to the templates, 5) building of a specific fragment libraryfor the query using FRAGFINDER, and 6) packing fragments and fold-recognition alignments to make a 3D structural model usingUNDERTAKER.

Energyminimization of the structural model provided by SAM-T06was carried out using the GROMOS96 implementation of Swiss PDBViewer [31]. Four hundred steps of steepest descent energy mini-mization were performed. The quality of the final model wasevaluated using WHAT IF (http://swift.cmbi.kun.nl/WIWWWI/)[32]. The molecular graphics software PyMol was used to visualizeand draw the structure [33].

Electrostatic potential calculations were performed solving thePoisson–Boltzmann equation with the DelPhi software package [34].The calculation was done using a protein and solvent dielectricconstants of 2 and 80, respectively, and a grid size of 240×240×240.The full charges scheme was taken from Klapper et al. [35].Representations of electrostatic maps were obtained with PyMol[33]. The calculation of the amino acid surface accessibilities weregenerated with Turbo Frodo software [36]. The pairwise sequencealignment between Hm EST and the best pdb hit, i.e., EST2, an esterasefrom the thermophilic eubacterium Alicyclobacillus acidocaldarius(pdb code: 1U4N, accession number: gi|55670177| [37]), was obtainedusing the SSM server, which generates protein structure alignmentsin three dimensions [38]. The alignment was drawn using ESPript2.0 [39].

2.5. Cloning of the lipC gene

H. marismortui was cultured in the Halobaculum gomorrensemedium, which consists of (g/L): NaCl, 125; MgCl2.6H2O, 160;K2SO4, 5; CaCl2.2H2O, 0.1; yeast extract 1; casamino acids 1 and starch2. The pHwas adjusted to 7.0 prior to autoclaving. Liquid cultures weregrown on an orbital shaker (160 rpm) at 37 °C. The genomic DNA fromH. marismortui was obtained by lysing the cell by osmotic shock inwater and extracting the DNA with phenol. The aqueous phase wasthen mixed with 2 volumes of cold ethanol and 0.1 volume of sodiumacetate 3M, incubated overnight at−20 °C and centrifuged for 10minat 10,000×g and 4 °C. The pellet was washed three times with ethanol80% (v/v) and solubilised in water.

A 984-bp fragment containing the lipC gene was amplified by PCRusing the H. marismortui genomic DNA as the template, Pfx DNApolymerase, the forward primer FwLipC (5′GTCGGGCATATGTCCACGA-CGGCCCGACC3′) and the reverse primer RevLipC (5′TCGACCGGATCCT-CAGAATCCGGGGCCGT3′). The underlined regions of these primersrepresent sites for the restriction enzymes NdeI (in the forwardprimer) and BamHI (in the reverse primer). The gene was amplified in30 cycles of 15 s at 94 °C, 30 s at 55 °C and 1 min at 68 °C. The PCRproduct was separated on a TAE-agarose 2% (m/v) gel, extracted usingthe PureLink™ Quick Gel Extraction kit of Invitrogen (San Diego, CA,USA) and digested with NdeI and BamHI. The digested product wasligated into pET14b, (previously digested with the same enzymes) togive the recombinant plasmid pET14b-lipC. The fragment cloned wassequenced with the T7 promoter and terminator primers on bothstrands. The recombinant plasmid pET14b-lipC had a nucleotidesequence coding for a His6-tag fusion in the N-terminal domain ofthe protein and a thrombin site between the 6 histidines and the firstcodon of the lipC gene.

2.6. Overexpression and protein purification

E. coli BL21(DE3)pLysS, transformed with pET14b-lipC, wascultivated in 1 L of Luria–Bertani (LB) medium to reach an OD600 of0.4. One mM IPTG (final concentration) was then added to the cultureto induce expression. The culture with IPTG was incubated for 4 hbefore harvesting of the cells by centrifugation (10,000×g for 5 min).

721M. Müller-Santos et al. / Biochimica et Biophysica Acta 1791 (2009) 719–729

The cell pellet was resuspended in 40 mL of lysis buffer (50 mM Tris–HCl, pH 8.0, 2.0 M KCl, 10 mM imidazole, 0.1% (m/v) Triton X-100).Cells were disrupted by ultrasonication (6 cycles of 10 s pulses, 90 W,with breaks of 10 s on ice) using a Branson Sonifier 450 (Danbury, CT,USA). The cellular debris was separated from the cell extract bycentrifugation at 15,000×g for 30 min. The supernatant was loaded(2.5 mL/min) onto a HiTrap Chelating column (5 mL) previouslyequilibratedwith 50mMTris–HCl, 2.0MKCl and 10mM imidazole, pH8.0. The His-tagged protein was then eluted with a stepwise additionof imidazole from 10 mM up to 500 mM in 50 mM Tris–HCl buffer pH8.0, containing 2.0 M KCl. The elution was done in four steps, 10, 50,200 and 500 mM of imidazole. Hm EST was eluted at 200 mM. Theeluted fractions were analyzed for enzymatic activity and by SDS-PAGE, as described below. The second step of purification was donewith an Octyl Sepharose CL-4B matrix. In this case 1 mL of gel wasplaced in a BioRad Poly-Prep column with gravity flow. The gel wasequilibrated with 50 mM Tris–HCl buffer, pH 8.0, 2.0 M KCl and 2.5 mgof protein was loaded per mL of gel. The protein was eluted with 5column volumes of 50 mM Tris–HCl buffer, pH 8.0, 2.0 M KCl. Underthese conditions the contaminants were retained on the hydrophobicgel while Hm EST was eluted. The eluted fractions were pooled andconcentrated by membrane filtration with Amicon® Ultra-15 10 Ktubes (Billerica, MA, USA). The purified protein was digested withthrombin from bovine plasma at 25 °C during 12 h (14.5 U/mg ofprotein) to release the N-terminal His6-tag. The thrombin wasseparated fromHm EST using gel filtration chromatography (Superose6 HR 10/30 in Tris–HCl 50 mM buffer and 2 M KCl; flow rate 0.3 mL/min). It was noted that the presence of His6-tag did not affectbiochemical properties of the enzyme (pH optimum, specific activityor substrate specificity). Therefore characterization using circulardichroismwas performedwith the Hm EST containing the His-Tag tail.

2.7. N-terminal sequence and mass determination

After proteolytic cleavage as described above, the recombinantprotein without the His6-tag was subjected to SDS-PAGE. Then thecleaved protein was transferred onto PVDF membrane and the bandcorresponding to the Hm EST was subjected to N-terminal sequenceanalysis using an Applied BiosystemModel 473A gas-phase sequencer.MALDI-TOFmass spectrometry was performed on Hm ESTwith the N-terminal His-Tag using Voyager DE-RP equipment (PerSeptiveBiosystems Inc., Framingham, MA, USA).

2.8. Electrophoresis and protein concentration determination

Protein concentration was measured either by the method ofBradford, using bovine serum albumin as the standard [40] or wascalculated from the OD value measured at 280 nm using the molarextinction coefficient (0.720 M−1 cm−1). Electrophoresis of proteinsamples was done by SDS-PAGE with polyacrylamide gels at 12% (w/v), and was performed according to the method of Laemmli [41]. Thegels were stained with Coomassie Brilliant Blue R-250 and destainedwith methanol/acetic-acid/water (5/1/4 v/v/v).

2.9. Circular dichroism

CD spectra of Hm EST in 10 mM sodium phosphate pH 8 and in thepresence of increasing KCl concentrations were recorded at 20 °C on aJasco 810 dichrograph (Jasco, Milano, Italy) using 1-mm thick quartzcells. CD spectra were measured between 200 and 260 nm, at 0.2 nm/min and were averaged from three independent acquisitions. Thecontribution of buffer was subtracted from experimental spectra.Spectra were smoothed using the “means-movement” smoothingprocedure implemented in the SpectraManager package. Meanellipticity values per residue ([Θ]) were calculated as [Θ]=3300×m×ΔA/(l×c×n), where m is the protein molecular mass

(36,168 Da), ΔA is the difference between right and left circularlypolarized lights, l is the path length (0.1 cm), c is the proteinconcentration (0.1 mg/mL) and n is the number of residues (349). Theexperimental data in the 200–260 nm range were analyzed using theDICHROWEB website (http://www.cryst.bbk.ac.uk/cdweb/html/home.html) which was supported by grants to the BBSRC Centre forProtein and Membrane Structure and Dynamics (CPMSD) [42,43]. TheK2d deconvolution method was used to estimate the α-helicalcontent.

2.10. Enzymatic activity measurements and kinetic characterization

Activity was determined by pH end-point titration using a pH-Stat(Metrohm, France). The reactions were done in a glass vesselthermostated at 37 °C containing 15 mL of 2.5 mM Tris–HCl, thesubstrate to be tested (see concentrations in Table 3) and various saltconcentrations. After addition of the enzyme, the free fatty acidsreleased during the reaction were titrated automatically with 0.1 MNaOH, with the amount of base added being recorded. One unit (U) ofenzymatic activity was defined as the liberation of 1 μmol of fatty acidper minute.

The influence of salt concentration on Hm EST activity wasanalyzed using different concentrations of NaCl (0–5.0 M) and KCl(0–4.0 M) and vinyl butyrate as the substrate. KCl was not used at 5 Mbecause it is not fully soluble at this concentration and at 37 °C. Fortesting substrate specificity, NaCl was used at a concentration of 2 M.The effect of pH was studied over the range of 4.0 to 9.0, using vinylbutyrate as the substrate. Since the pKa value of butyric acid is 4.8, themeasurements of esterase activity at low pH values were performedby back titration to pH 9, after a reaction period of 3 min at the pHvalue of the assay.When back titrations were performed, for each pH ablank assay was performed without enzyme to determine the amountof NaOH required to increase the pH to 9 in the absence of fatty acidrelease. This value was subtracted from the back titration value.

The thermostability was evaluated by incubating the enzyme(0.1 mg/mL) at 40, 50 and 60 °C during 2 h, and then determining theresidual activity at 37 °C, in 2.5 mM Tris–HCl buffer, pH 8.5, 2 M NaCl,with vinyl butyrate as the substrate.

2.11. Reactivation assay

The enzyme (1 mg/mL) was dialyzed against 50 mM Tris–HClbuffer pH 8.0 without salt, during 16 h and the residual activity wasmeasured. The dialyzed enzyme was then mixed with KCl to yield asalt concentration of either 2 or 3 M. These solutions were incubatedat 4 °C during 16 h and the activities of samples removed during thisperiod were determined at 37 °C using a pH-Stat in 2.5 mM Tris–HClbuffer at pH 8.5, with vinyl butyrate as substrate and 2 M NaCl.

2.12. Inhibition assay

Inhibition of Hm EST was tested with the oxadiazolone compound7600 (5-methoxy-3-(4-phenoxyphenyl)-3H-[1,3,4]oxadiazol-2-one),a molecule previously described as being a specific inhibitor of themembers of the HSL family [44]. Compound 7600 was a generous giftfrom Dr. Stefan Petry (Sanofi-Aventis, Frankfurt am Main, Germany).These assays were done in aqueous medium and in the absence ofsubstrate [45]. An Hm EST solution was prepared in 50 mM Tris–HClbuffer, pH 8.0, 2 M KCl, at a protein concentration of 0.38 mg/mL(11 μM). A 10 mM stock solution of compound 7600 was prepared indimethyl sulfoxide (DMSO) and a 20 μL aliquot was added to 180 μL ofthe Hm EST solution, thereby giving a solution containing 1 mM of7600 and 10 μM of Hm EST (i.e. an inhibitor to enzyme molar ratio of100:1) and 10% DMSO. The residual activity was then measured atvarious incubation times, using vinyl butyrate as the substrate understandard conditions (Tris–HCl 2.5 mM, pH 8.0, 2 M NaCl). A control

Fig. 1. Alignment of the amino acid sequence of Hm EST with other proteins of the HSLfamily showing the (A) oxyanion hole loop and (B) the pentapeptide containing theactive site serine. H. marismortui esterase (Hm EST), esterase from Alicyclobacillusacidocaldarius (EST2), Archaeoglobus fulgidus esterase (Af EST), Mycobacteriumtuberculosis esterase (Rv1339c), Bacillus subtilis Brefeldin A esterase (Brefeldin A),Rhodococcus sp. heroin esterase (Rh EST), Streptomyces viridochromogenes acetylhydrolase (Sv AH), rat hormone sensitive lipase (rHSL) and human hormone sensitivelipase (hHSL). The residues marked in bold indicate the most conserved residues withinthe amino acid sequences. The catalytic serine is marked (⁎).

722 M. Müller-Santos et al. / Biochimica et Biophysica Acta 1791 (2009) 719–729

without 7600 but with 10% DMSO gave no loss of activity. Inhibition bydiethyl-p-nitrophenyl phosphate (E600) and phenylmethylsulfonylfluoride (PMSF) were evaluated in a similar manner, except that theinhibitor:enzyme molar ratio was 10:1.

3. Results

3.1. Selection of Hm EST

Our search of available haloarchaea genomes identified the lipCgene of H. marismortui as coding for a putative lipase or esterase (HmEST). A BLAST search showed that the sequence of this gene had arelatively low identity with the most closely related sequencesdeposited in GenBank (Table 1). These sequences code for putativeesterases or lipases, but they only had 32–39% amino acid identitywith the Hm EST sequence. An interesting feature identified by thisanalysis was a low estimated pI of 4.24, this being due to a relativelyhigh number of acidic residues (aspartic and glutamic acid), whichaccount for 16.8% of the amino acids in the sequence. Neithertransmembrane domains nor a signal peptide were identified,indicating that Hm EST is probably a cytosolic enzyme.

The conserved esterase/lipase domain identified by the in silicoanalysis of Hm EST is an α/β hydrolase domain that is common toesterases/lipases/thioesterases [46]. Within this domain, Hm ESTcontains two regions that are common to esterases/lipases, namelythe G-X-S-X-G pentapeptide, covering residues 126–130 and contain-ing the active site serine, and the oxyanion hole loop, spanningresidues 55 and 58. Hm EST was also found to be homologous toseveral esterases/lipases from the Hormone Sensitive Lipase (HSL)family (Fig. 1). The degree of similarity suggests that Hm EST belongsto this family, although some differences are observed in both theoxyanion and the pentapeptide regions. With respect to the oxyanionhole loop, the sequence HGGG is fully conserved among the othermembers of the HSL family whereas the sequence RGGA is found inHm EST. With respect to the pentapeptide domain, the second residueof Hm EST is a histidine (position 127) whereas other HSL proteinsnormally contain an aspartate or glutamate residue in this position.

3.2. Cloning, overexpression and purification of Hm EST

After purification of the genomic DNA of H. marismortui, the lipCgene coding for Hm EST was amplified by PCR and cloned into theplasmid pET14b. The recombinant proteinwas overexpressed in E. coliBL21(DE3)pLysS, giving about 10mgof pureHmEST per litre of culturebroth. The results of the purification are summarised in Table 2. Afterthe IMAC step the protein content decreased significantly, but thespecific activity increased 14-fold. One hypothesis to explain this gainin activity is that various compounds present in cell lysate inhibit theenzyme until they are removed by the first purification step. Anotherexplanationmight be that the protein is unfoldedwithin the cytoplasmof the E. coli host and begins folding correctly only once it is liberated

Table 1Bioinformatic analysis of the Hm EST (LipC) sequence and related sequences found by BLAS

Gene name GeneIDa Domain organism M.M. (kDa)b Ident

lipC 3128745 Archaea Haloarcula marismortui ATCC 43049 34004.1 100ZP_01736589 Bacteria Marinobacter sp. ELB17 32890.3 39ABX10578 Bacteria, uncultured planctomycete 5H12

(environmental sample)38510.1 32

ZP_01094419 Bacteria Blastopirellula marina DSM 3645 29547.0 35YP_001544283 Bacteria Herpetosiphon aurantiacus ATCC 23779 41954.5 32

a Gene identification and/or name available in the NCBI (GenBank).b Molecular weight calculated by the ProtParam tool (ExPASy) based on the amino acid sc Identity results obtained by the protein BLAST search when the lipC of H. marismortui isd Number of negatively charged residues, aspartate and glutamate, calculated by the Proe pI calculated by the ProtParam tool (ExPASy) based on the amino acid sequence.

into the 2 M KCl lysing solution. If folding is a slow process, then itmight be still going on during the IMAC purification step. The N-terminal His6-tag was subsequently eliminated by thrombin cleavagewithout variation of specific activity of Hm EST.

An interesting observation was the aberrant migration of recom-binant Hm EST upon separation by SDS-PAGE. The migration of HmEST was consistent with that of a protein of around 50 kDa (Fig. 2),whereas the theoretical molecular mass of Hm EST was estimated tobe 34 kDa and theMALDI-TOF analysis of the proteinwith the His6-taggave a value of 36,167.5 Da. The protein band appearing at 50 kDa on

T search.

ityc (%) Annotation (Asp+Glu)d % (Asp+Glu)/totalof amino acids

Theoretical pIe

Putative lipase/esterase 55 16.8 4.24Probable lipase/esterase 20 6.5 7.17Secreted lipase/esterase 43 12.3 6.50

Probable lipase/esterase 23 8.3 6.49Esterase/lipase-like 29 7.4 5.60

equence.the query sequence.

tParam tool (ExPASy).

Table 2Purification of Hm EST.

Step Volume(mL)

Total protein(mg)

Total activity(U)

Specific activity(U/mg)

Purificationfactor (fold)

Yield ofactivity (%)

Lysate 36 324 6750 21 1.0 100Immobilized Metal Affinity Chromatography (IMAC) 32 32 9200 288 14 136Hydrophobic interaction chromatographyand concentration

2.5 9.5 5415 570 27.5 80

Purification after proteolytic cleavage 2.5 8.3 4725 570 27.5 70

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the gel was transferred onto PVDFmembrane, subjected to N-terminalsequencing and identified as Hm EST. It is worth noticing that thisaberrant migration is characteristic of halophilic proteins andprobably occurs due to the unusually high proportion of negativelycharged residues (Asp and Glu) present in these proteins [47]. Similarresults were obtained in nondenaturing and nonreducing conditionssuggesting that the aberrant migration was not due to an aggregationof the protein. Indeed, a dimer should migrate around 68 kDa, notclose to 50 kDa. Actually, this phenomenon could be due to theproteins bindingmuch less SDS (as compared to nonhalophilic proteinmarkers), thus making the SDS-protein complex less mobile, and hasbeen already described in other purified proteins from halophilicarchaea [48].

3.3. Salt dependence of Hm EST activity

As preliminary experiments indicated a complete lack of activitywhen Hm EST was assayed in the absence of salt, the effect of saltconcentration on the enzyme activity was studied using either KCl orNaCl with concentrations ranging from 0 to 4 M, and from 0 to 5 M,respectively (Fig. 3). Although the enzyme sample contained 2 M KCl,the salt contributed by the enzyme in the assay was negligible, as thevolume of the enzyme (10 μL) that was added to the reaction mixturewas negligible with respect to the final volume of this latter (15mL). Aclear salt-dependent activity was observed, with no activity beingdetectable in the absence of salt, and with the maximum specificactivity being reached at either 2 M NaCl (590 U/mg) or 3 M KCl(634 U/mg). Further increasing the salt concentration resulted in adecrease in the specific activity, where this latter was reduced toeither 74% or 80% of the maximum value depending on whether NaCl5 M or 4 M KCl was added, respectively. The lack of activity in theabsence of salt could be accounted for by Hm EST denaturation at low

Fig. 2. SDS-PAGE analysis of Hm EST. Protein samples were loaded onto an SDS-PAGE(12%) gel under reducing conditions. The proteins were stained with Coomassiebrilliant blue R250. Lane 1, low molecular weight standards; lane 2, pool of fractionscontaining Hm EST after IMAC chromatography (20 μg); lane 3, purified Hm EST afterhydrophobic interaction chromatography (20 μg).

salt concentration. To check this hypothesis, the salt was completelyremoved by dialysis and no residual activity was detected after thisstep. Then, KCl was added to the protein to final concentrations of0.25, 0.5, 2 or 3 M. A low activity was detected when the protein wasincubated in 0.25 (20 U/mg) of 0.5 M (90U/mg) of KCl. However uponaddition of either 2 M or 3 M of KCl, a significant recovery of activitywas observed, with this recovery occurring in a time-dependentmanner (Fig. 4). The maximum recovered activity did not reachhowever the pre-dialysis value of 590 U/mg. Indeed, in the presenceof KCl 2 M, the activity reached 58% of this value, while with 3 M KClonly 40% of the pre-dialysis activity was recovered. For both KClconcentrations, the recovery of activity over the first 2 h could bereasonably approximated by a first order model, with a half-reactivation time of approximately 54 min.

3.4. Salt-dependence of Hm EST folding

To assess whether the results described above were related to thedegree of folding of Hm EST, we recorded far–UV circular dichroism(CD) spectra of the enzyme, both in the absence and in the presence ofincreasing concentrations of KCl (0, 0.25, 0.5 and 2 M). Although KF isin principle to be preferred because fluoride does not absorb light inthe far–UV range, contrary to chloride, the latter was chosen (i) for thepurpose of a direct comparisonwith biochemical and kinetic data thatwere all collected in the presence of KCl and (ii) in order to betterreflect the physiological environment of the enzyme, where chlorideions are largely prevalent [14,15]. The far–UV CD spectrum of Hm ESTin the absence of salt is typical of an unfolded protein, as shown by thelargely negative ellipticity at 200 nm and by the modestly negativeellipticity in the 210–225 nm region (Fig. 5). Although the addition ofincreasing concentrations of KCl caused a dramatic drop in the signalto noise ratio, awell-knownphenomenon that is due to the absorption

Fig. 3. Effect of the NaCl and KCl concentration on the activity of Hm EST. The assays todetermine the enzymatic activity were carried out at 37 °C in 2.5 mM Tris–HCl, pH 8.5with 73.6 mM of vinyl butyrate, using the pH-Stat technique. A blank assay without theenzyme was performed to determine the spontaneous hydrolysis rate of the substrate.Values are means±SD (n=3).

Fig. 4. Time-dependent reactivation of Hm EST upon salt addition. Two KClconcentrations were evaluated for the reactivation assay: 2 M and 3 M KCl. Therecovery of enzyme activity during the incubation at 4 °C was determined by thetitrimetric method, at 37 °C in 2.5 mM Tris–HCl, pH 8.5 with 73.6 mM of vinyl butyrateand 2 M of NaCl. Values are means±SD (n=3).

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of light by chloride ions, analysis of the CD spectra pointed to anincrease in overall α-helical content, as illustrated by the increase inthe ellipticity at 200 nm and by the more pronounced negativeellipticity in the 210–225 nm region (Fig. 5). Such an increase in α-helicity was also confirmed upon deconvolution of the CD spectra andquantitative estimation of the secondary structure content, whichpointed to an increase from 7% (0M KCl) to 9% (500mMKCl) in theα-helical content. It should be pointed out, however, that at these KClconcentrations, the protein still remains predominantly unfolded, asseen by the largely negative ellipticity at 200 nm.

This salt-dependent folding propensity of Hm EST was furtherconfirmed in the presence of 2 M KCl, albeit the CD spectrumdisplayed a very poor signal to noise ratio due to strong KCl absorption(data not shown). These results confirm that the lack of activity in theabsence of salt is due to a total denaturation of the enzyme and thatincreasing the salt concentration triggers protein folding. Accordingly,

Fig. 5. Far–UV CD spectra of Hm EST (0.1 mg/mL) in 10mM sodium phosphate buffer pH 8 inmg) and 500 mM (Specific activity 20 U/mg)). Each spectrum is the mean of three indepen

the predominantly unfolded nature of Hm EST at 0.5 M KCl wouldexplain the enzymatic activity data, which showed that only 50% ofthe maximum activity was observed at this salt concentration (Fig. 3).

3.5. Effect of pH on Hm EST activity

Hm EST was not active on vinyl butyrate at pH values below 5.0(Fig. S1). Above pH 5.0 the specific activity increased with pH,reaching a maximum of 590 U/mg at pH 8.5. At pH 9, the specificactivity began to fall off, being 78% of the maximum value. Values ofpH above 9 were not tested due to a spontaneous hydrolysis of vinylbutyrate.

3.6. Thermostability of Hm EST

Hm EST, at a concentration of 0.1 mg/mL, was incubated for 2 h at40, 50 and 60 °C, at pH 8.0 with 2MKCl. Therewas no loss of activity at40 °C, only a 4.5% loss of activity following incubation at 50 °C, but a100% loss of activity following incubation at 60 °C. The loss of activityat 60 °C was monitored over time, and all activity was lost within5 min.

3.7. Substrate specificity of Hm EST

The activity of Hm EST was tested against various vinyl esters andtri-, di- and monoacylglycerols, in the presence of 2 M NaCl and at pH8.5 (Table 3). The highest specific activity (590 U/mg) was measuredwith vinyl butyrate, which was used for the standard assay of Hm ESTthroughout this study. High specific activities were also measuredwith other short chain vinyl esters (vinyl acetate, propionate, andhexanoate). The activity of Hm EST on vinyl esters with longer acylchains was much lower. The specific activities on tri-, di- andmonoacylglycerols were much lower than those measured with theshorter-chain vinyl esters and decreased with increasing acyl chainlength. There was no detectable activity of Hm EST on vinyl esters andglycerol esters that have acyl chains longer than 12 carbon atoms.

The degree of glycerol esterification also had an important impacton the specific activity of HmEST. For example, the specific activities ofHm EST on dibutyrin and monobutyrin were 13-fold and 26-foldgreater, respectively, than the specific activity on tributyrin (Table 3).

the presence of increasing concentrations of KCl (0 mM, 250 mM (Specific activity 90 U/dent acquisitions.

Table 3Substrate preferences of Hm EST.

Substrates (mM/SL)d Specific activity(U/mg)

% Maximalactivityc

Vinyl estersVinyl acetatea (330/315) 180±0.5 30.5Vinyl propionate (86.6/82) 325±35.4 55Vinyl butyrate (73.6/22) 590±0 100Vinyl hexanoate (58.4/I) 400±0 67.8Vinyl octanoate (48.2/I) 40±0 6.8Vinyl decanoate (41.4/I) 12.8±0.4 2.2Vinyl laurate (35.9/I) 3.2±0.3 0.54

Triacylglycerolsb

Triacetin (320/293) 32±0.5 5.4Tripropionin (41/12) 20±0 3.4Tributyrin (34/0.8) 5.8±0.6 0.98Tricaprylin (20/I) 2.2±0 0.37Tricaprin (6/I) 1.3±0 0.22

DiacylglycerolsDibutyrin (45.3/42) 74.4±11.7 12.6Dicaprylin (9.6/I) 2.5±0 0.42Dicaprin (8.3/I) 1.1±0.2 0.19

MonoacylglycerolsMonobutyrin (190/164) 151.3±0.3 25.7Monocaprylin (15.3/12.5) 26.9±2.9 4.6Monocaprin (13.5/I) 4.8±0.2 0.8Monolaurin (12.2/I) 1.8±0.2 0.3

Experiments were performed with vinyl esters and tri-, di- and monoacylglycerols at37 °C in 2.5 mM Tris–HCl (pH 8.5) buffer, 2 M NaCl with stirring. The number betweenparentheses after the substrate name corresponds to the concentration in the assay(mM).

a Assays with vinyl acetate were performed in the same buffer at pH 7 to avoidchemical hydrolysis at pH 8.

b Activities with glycerol esters were determined under the same experimentalconditions as those for vinyl esters but with 1% Triton® X-100 (final concentration). 1 Ucorresponds to 1 μmol of fatty acid released per min.

c The percentage of the maximal activity was calculated using the activity againstvinyl butyrate as 100%.

d All the substrates were assayed at concentrations that were above their solubilitylimits.

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3.8. Effects of serine esterase inhibitors on Hm EST activity

Three serine esterase inhibitors were tested against Hm EST (Fig.S2): PMSF, E600, and compound 7600, the last of these inhibitorsbeing described as a specific inhibitor of enzymes belonging to the HSLfamily [44]. Inhibition of Hm EST by PMSF and E600 was fast, withhalf-inactivation times of 10 s and 20 s, respectively, and no residualactivity being detected after 90 s (Fig. S2 A). Hm EST inhibition bycompound 7600 was much slower, with a half-inactivation time of15 min and a total loss of activity after 85 min (Fig. S2B). Inhibition byPMSF, E600 and 7600 indicate that Hm EST is a serine enzyme, as hadbeen suggested by the sequence alignment. Furthermore, the fastinhibition of Hm EST by PMSF and E600 in the absence of detergentssuggests that Hm EST has an active site accessible to the solvent, astructural feature that usually differentiates esterases from lipases.Finally, inhibition by compound 7600 indicates that Hm EST belongsto the HSL family, although this inhibition is weaker than thatobserved with other enzymes of this family [44].

3.9. Structural modeling and analysis of Hm EST

In order to obtain structural insights into Hm EST, we generated a3D structural model using the SAM-T06 server. The 10 best structuralalignments, as provided by the server, include 9 esterases and onepeptidase (pdb code 2HU5), with E-values ranging from 5.6E−33 forthe best hit (Fig. 6 A) to 4.45E−19 for the most distant hit (Table S1).The final model (Fig. 6 B) covers the entire amino acid sequence of theprotein, and very well superimposes onto that of the carboxylesterase

EST2 (pdb code 1U4N) [37], with an rmsd of 1.65 Å over 236 Cα atoms(Fig. 6 C).

Notably, a threading approach using the Phyre server [49] led to amodel that was based on the structure of EST2 with an E-value of1E−33 and an estimated precision of 100% (data not shown). Thethreading model, which encompasses residues 10 to 279, was nicelysuperimposable onto that provided by the SAM-T06 server (rmsd of1.71 Å over 224 Cα atoms) (data not shown). Although the modelgenerated by the SAM-T06 server possesses a long C-terminalextension with respect to the Phyre model, the two models sharethe same overall central topology, despite minor structural differenceswithin loops (data not shown). The quite good agreement betweenthe twomodels points out the reliability of both structural predictions.

The overall quality of the model provided by the SAM-T06 serverwas assessed through evaluation of the Ramachandran plot. To thisendeavor, we used the corresponding analysis implemented withinWHAT IF [32]. This server analyzes the Ramachandran plot of a PDBfile and compares it with the Ramachandran plots of about 400representative structures solved at high resolution. The Ramachan-dran Z-score (−2.644), as provided by WHAT IF [32], is within theexpected range for well-refined structures, and the Ramachandranplot (Fig. S3) points out the overall good quality of the model, withonly two residues (Thr175 and Asp30) and the catalytic serine(Ser128), lying in an unfavorable region of the plot.

The final model for Hm EST consists of eleven α-helices and acentral β-sheet core containing eight β-strands (Fig. 6 B). The putativeoxyanion loop of Hm EST (residues 55–58, Fig. 1) superimposes wellonto the oxyanion hole loop of EST2 (Fig. 6 D). In other words, thebackbones of residues Gly56, Gly57 and Ala58 are found to be close tothe active site and they might be involved in the stabilization of thereaction intermediate. Residues Ser128, Asp224 and His254 of Hm ESTsuperimpose well onto the residues forming the catalytic triad of EST2(Ser155, Asp252 and His282) (Fig. 6 D).

Althoughwe are quite confident in the overall reliability of the coreof the structural model of Hm EST, the additional C-terminal α-helix(residues 297–313) should be considered with caution. Indeed the180–327 region of Hm EST is predicted to be disordered, as judged byusing the Metaserver of Disorder, MeDor [50] (data not shown).Furthermore, it systematically represents an additional structuralelement with respect to the best-scoring structural templatesprovided by both the SAM-T06 and the Phyre server (data notshown). Hence, coordinates for this region were generated by theSAM-T06 server through fold recognition methods of distantly relatedstructures followed by combination of the generated fragments.Therefore, it should be noted that the proposed structure for thisregion is merely tentative and that this region may adopt a quitedifferent fold and may occur in a different spatial position. Never-theless, given its flexible nature, this region is very likely to be solvent-exposed: as reported by Liu et al., long regions of NOn RegularSecondary Structure (NORS) are generally solvent accessible [51]. Assuch, irrespective of its actual conformation, this region probably doescontribute to the protein surface properties.

The distribution of charges of Hm EST was estimated from thecalculation of the electrostatic potential of the model (Fig. 7 A, top andbottom). Hm EST shows a high negative potential, mainly located onthe surface, while the electrostatic potential of EST2 is 1.5-fold lower(Fig. 7 B, top and bottom). The high negative potential of Hm ESTprobably results from the presence of clusters of aspartate andglutamate residues, while no clusters of positive charges wereobserved. The analysis of the electrostatic surface showed that mostacidic residues are located at the protein surface, being thus exposedto the solvent. This enrichment in acidic residues at the protein surfacemay favor solvation, as already observed for the glucose dehydrogen-ase from Haloferax mediterranei, which has an important arrangementof water molecules interacting with solvent-exposed glutamate andaspartate residues [52]. In the same vein, it has been observed that the

Fig. 6. (A) Pairwise sequence alignment between Hm ESTand EST2 that share 20% identity. Identical amino acids are boxed in red, while similar residues are drawn in red. The dots inthe alignment and in the structural elements indicate gaps. The secondary structure elements of the Hm EST model and of the EST2 crystal structure (pdb code: 1U4N) are shownabove and under the alignment, respectively. The numbers at the beginning of each line correspond to the amino acid positions. Dots above the alignment indicate intervals of 10residues. The residues building up the catalytic triad aremarked with asterisks, while the oxyanion hole loop is underlined in red. (B) Cartoon representation of the model of Hm ESTas obtained by using software programs from the SAM-T06 server. α-helices are shown in orange, β-strands in blue and the active site residues in red are shown in CPK.(C) Superimposition between themodel of Hm EST (green) and the crystal structure of EST2 (pink). (D) Superimposition of the residues of the oxyanion hole and of the catalytic triadfrom Hm EST and EST2. Colors are as in panel C.

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solvation degree of the glucose dehydrogenase from H. mediterraneiis among the highest values occurring in proteins of known 3Dstructure. Surprisingly, only five potassium ions were observed tointeract with this latter protein, which clearly points to a role for thesolvent-exposed acidic residues in solvating the protein in a

desiccating condition, rather than in establishing direct interactionswith ions.

Hm EST and EST2 are both characterized by a high and similarcontent of acidic residues (Asp+Glu; 16.82% and 13.23%, respectively;Table 4). This content is much higher than that of globular proteins of

Fig. 7. (A, B) Relative surface electrostatic potentials of Hm EST (A) and of EST2 (B) as obtained using the DelPhi software program (see Materials and methods section). Thestructures are graphically depicted with the active site cleft facing up (first row) or as the 180° rotated view (second row), with the red surface corresponding to negatively chargedresidues (Asp, Glu) and the blue surface corresponding to positively charged residues (Arg, Lys).

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known 3D structures (11.76%; see Table 4). Hm EST differs, however,from EST2 and other globular proteins in its low content of basicresidues (5.81% for Hm EST versus 8.71% for EST2). The acid to basicresidue ratio in Hm EST (2.89) is therefore much higher than that ofEST2 (1.52) and other globular proteins (1.1; Table 4).

The charged to hydrophobic residue ratios of Hm EST (0.48) andEST2 (0.40) are similar to that of globular proteins (0.50) on average.The accessible hydrophobic surface of Hm EST (31.73% of the totalaccessible surface of the protein) is, however, lower than that of EST2(38.64%). The charged to hydrophobic surface ratio is thus higher inHm EST (1.36) than in EST2 (1.07).

4. Discussion

4.1. The first biochemical characterization of a halophilic esterase

Although the bacterial expression and characterization of severalesterases originating from extremophiles, such as the thermophilePyrococcus furiosus [8], the thermoacidophilic Sulfolobus solfataricus[9,12], the hyperthermophile Aeropyrum pernix K1 [10], Archaeoglobusfulgidus [11] and Alicyclobacillus acidocaldarius [37], were previouslyreported, this article reports the first biochemical characterization ofan esterase from a halophilic archaea. However, other esterasesexhibiting better activity at high salt concentration have been reportedin a metagenomic study from the brine:seawater interface of theUrania hypersaline basin [53]. This basin in the Eastern Mediterraneanis a deep, anoxic, high pressure and hypersaline environment [54].

Hm EST shows a preference for esters containing short-chain fattyacids and for monoesters and its inhibition by E600 and PMSF supportthe identification of Hm EST as a serine carboxylesterase. Moreover,the sequence alignments indicate that Hm EST belongs to the HSLgene family, like two other previously characterized archaealesterases, namely Af EST from A. fulgidus [11] and Sso ESTfrom S.solfataricus [9]. Supporting these findings, Hm EST is inhibited by

compound 7600, an inhibitor with a high specificity for the esterasesand lipases of the HSL gene family [44].

4.2. Amino acid composition and surface properties of Hm EST

Halophilic proteins are characterized by a high negative surfaceelectrostatic potential, primarily due to the high abundance of acidicresidues at the surface of the protein. A high surface density ofnegative charges is believed to stabilize the structure of halophilicproteins in the high salt concentrations that exist in the cytoplasm ofhalophilic archaea [14–16]. Indeed, halophilic adaptation is bestexplained by the solvation–stabilization model, whereby surfaceacidic residues bind hydrated ions to form a solvation shell thatprevents water or salt enrichment at the protein surface, thus allowingthe proteins to remain soluble and properly folded at high saltconcentration [14,15,55]. This model is supported by the fact thatproteins fromhalophilic archaea have, on average, a higher percentageof Asp and Glu residues exposed at the surface than do proteins fromthermophilic archaea [56].

As expected, the 3D model of Hm EST shows a high density ofacidic residues exposed at the surface of the protein (28.06% of thetotal accessible surface versus 24.02% in EST2, Table 4). Notably, thehigh acidic content of Hm EST also provides an explanation for itslow pI value and the atypical electrophoretic migration of theprotein (Fig. 2). It is noteworthy that the proteins identified by theBLAST search as having the highest amino acid identities with HmEST are putative lipases/esterases of marine bacteria. These micro-organisms are adapted to sea water, an environment with salinity of3.5% (w/v), and are classified as moderate halophiles [57]. Asequence analysis indicates that Hm EST has almost a 2-fold higherproportion of acidic residues (Asp+Glu) than the putativeesterases/lipases of most of these bacteria. This finding confirmsthat the proportion of acidic residues is positively correlated withthe degree of halophilicity.

Table 4Amino acid and accessible surface analysis of Hm EST and EST2.

Hm EST EST2 Globularproteins [61]

Amino acid frequenciesAsp+Glu (% of total residues) 16.82% 13.23% 11.76%Arg+Lys (% of total residues) 5.81% 8.71% 10.66%(Asp+Glu)/(Arg+Lys) ratio 2.89 1.52 1.10Hydrophobic residues (% of total residues) 47.40% 54.19% 44.73%Charged/hydrophobic residue ratio 0.48 0.40 0.50

Amino acid surface accessibilitiesTotal accessible surface (Å2) 15,632 13,611Accessible surface of Asp+Glu (Å2) 4387 3273Asp+Glu (% of total surface) 28.06% 24.05%Accessible surface of Arg+Lys (Å2) 2342 2354Arg+Lys (% of total surface) 14.98% 17.29%(Asp+Glu)/(Arg+Lys) surface ratio 1.87 1.39Accessible surface of hydrophobic residues 4960 5259Hydrophobic residues (% of total surface) 31.73% 38.64%Charged/hydrophobic surface ratio 1.36 1.07

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Beyond the difference in the composition of acidic residues, HmEST and EST2 also differ in the density of basic residues accessible attheir respective surfaces, this density being much lower in Hm EST(14.98%) than in EST2 (17.29%). As a result, the overall electrostaticpotentials of Hm EST and EST2 are very different (Fig. 7). This findingstrongly supports an important role of the ratio between accessiblebasic and acidic residues in halophilic adaptation. Finally, the C-terminal extension of Hm EST may also play a role in halophilicadaptation by serving as a stabilizing factor in high salt, as alreadyreported for other halophilic enzymes [55].

4.3. The effect of salt concentration on the esterase activity of Hm EST

The phenomenon of enzyme inactivation at low salt concentrationsand activation at high salt concentrations that was shown herewith HmEST has not been previously reported for an esterase. This phenomenonhas been, however, observed for other halophilic enzymes, such as H.marismortui malate dehydrogenase [15], H. mediterranei glucosedehydrogenase [58],Halobacteriumsalinarumglutamatedehydrogenase[59] and Natrinema sp. protease [60]. Mevarech et al. reported that thereactivation of malate dehydrogenase from H. marismortui wasnegatively correlated with the incubation time at low salt concentra-tions [15]. In our case, Hm EST was dialyzed for 16 h to remove saltsbefore the reactivation/refolding experiments were performed byadding various amounts of salts. This long incubation time at low saltconcentrations might explain why we did not recover 100% of Hm ESTactivity after addition of 2MKCl. Our results are similar to those of Shi etal. [60]: when they removed the salt from the protease of Natrinema sp.by dialyzing the sample during 9 h, reintroduction of NaCl to 2.5M gaveonly 60% of the pre-dialysis activity. We advance the hypothesis thatthese results are due to a two-step denaturationprocess: in the first stepthe proteinwould lose its secondary structure but remain in a state thatcan refold in a reversible way if salt is added and does so, with a half-lifeof around 1 h. In the second step, however, while being maintained atlow salt concentrations, the protein would slowly and irreversibly passto a new conformation.

In conclusion, this study provides the first description of anesterase showing a specific salt-dependency of its enzyme activitythrough a salt-dependent folding. It also provides new insights forunderstanding the structure–function relationships of halophilicenzymes in general.

Acknowledgements

The French–Brazilian cooperation was made possible by a grantfrom CAPES-COFECUB. The experimental work performed at the EIPL

laboratory was supported by the Centre National de la RechercheScientifique (CNRS). Marcelo Müller-Santos, David Mitchell and NadiaKrieger thank CNPq (Conselho Nacional de Desenvolvimento Cientí-fico e Tecnológico), a Brazilian Government Agency for the advance-ment of science and technology, for research scholarships. The authorsalso thank Prof. Francisco Rodriguez-Valera and Arantxa Lopez-Lopez,from Universidad Miguel Hernandez, Alicante, Spain, for protocols onHaloarcula marismortui cultivation. We thank Stéphanie Costanzo(AFMB laboratory, Marseille) for recording the CD spectra, and RegineLebrun (IBSM, Marseille) for performing N-terminal sequencing andMALDI-TOF analysis.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.bbalip.2009.03.006.

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